Cartilage gel for cartilage repair, comprising chitosan and chondrocytes

ABSTRACT

The present invention concerns a method for obtaining an implantable cartilage gel for tissue repair of hyaline cartilage, comprising particles of chitosan hydrogel and cells that are capable of forming hyaline cartilage, said method comprising a step for amplification of primary cells in a three-dimensional structure comprising particles of physical hydrogel of chitosan or a chitosan derivative, then a step for re-differentiation and induction of the synthesis of extracellular matrix by said amplified cells, in the same three-dimensional structure, wherein said cells are primary articular chondrocytes and/or mesenchymal stem cells differentiated into chondrocytes. The present invention also concerns the cartilage gel obtained thereby, and its various uses for cartilage repair following a traumatic lesion or an osteoarticular disease such as osteoarthritis. The invention also concerns a three-dimensional matrix comprising particles of physical hydrogel of chitosan or of chitosan derivative, optionally supplemented with an anionic molecule such as hyaluronic acid or a derivative of hyaluronic acid or a complex of hyaluronic acid.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of of copending application Ser. No.15/532,020, filed on May 31, 2017, which is the National Phase under 35U.S.C. § 371 of International Application No. PCT/FR2015/053271, filedon Dec. 1, 2015, which claims the benefit under 35 U.S.C. § 119(a) toPatent Application No. 1461746, filed in France on Dec. 1, 2014, all ofwhich are hereby expressly incorporated by reference into the presentapplication.

The present invention relates to compositions, allowing inter alia thereconstruction of cartilage, and to a method for obtaining suchcompositions. More particularly, the present application relates to anenvironment or scaffold which is extremely favourable not only for theproliferation of cells that are capable of forming hyaline cartilage,but also for the synthesis of cartilage extracellular matrix by thesecells; the cells in this environment or scaffold constitute animplantable composition which can be implanted to cartilage defects inhumans or animals. The structure also constitutes a favourableenvironment during implantation.

Cartilage, or cartilaginous tissue, is constituted by specific cells,namely chondrocytes, distributed in an extracellular matrix, comprisingat least 80% water. The chondrocytes are capable of synthesizing ordegrading components of the cartilage extracellular matrix, composed ofglycosaminoglycans and collagen fibers, essentially type II for hyalinecartilage. Thus, the chondrocytes are responsible not only forsynthesis, but also for maintaining cartilaginous tissue.

Cartilage, and particularly articular cartilage in the adult, has a verypoor self-repair capacity, primarily because of its avascular nature andbecause mature chondrocytes do not proliferate. Thus, cartilaginouslesions are essentially irreversible, and as a result constitute a majorcause of pain and disability, in particular as a consequence of trauma,mechanical wear or a degenerative articular disease such asosteoarthritis. There are currently no entirely satisfactory therapeuticsolutions that can be used to treat cartilaginous defects, andparticularly large defects.

Various surgical procedures have been tested, consisting either offilling the lesion with materials intended to mimic the elastic andcompressivity properties of natural cartilage, or implanting cells, inparticular chondrocytes, with the aim that they will synthesizeextracellular matrix de novo and thus repair the defect. A morepromising approach is based on the implantation of cartilaginous tissue,i.e. a neo-tissue obtained from chondrocytes. Implantation of autologouschondrocytes (“ACI”, autologous chondrocyte implantation) has now beenused for several decades to treat patients suffering from cartilaginousdefects. However, a major difficulty with chondrocytes implantation, andin particular autologous chondrocytes, resides in the number of cells tobe implanted. In fact, in general, only a small number of chondrocytescan be removed from the native cartilage of the patient to be treated;as a consequence, it is necessary to carry out a first step ofamplifying the chondrocytes removed in order to drastically increasetheir number. However, in vitro amplification of chondrocytes until asufficient number of cells for implantation has proved to be verydifficult. During this amplification step, it is in fact known that thechondrocytes dedifferentiate and lose their chondrocyte phenotype, thenexpressing type I collagen rather than type II collagen, such that whenre-implanted, they do not lead to the formation of a tissue withsatisfactory properties, but rather to a scar style tissue or, in thecase of a cartilage repair, to the formation of a non-functionalfibrocartilage, essentially composed of type I collagen. Some authorshave proposed culture media which allow the chondrocytes which havededifferentiated during the monolayer proliferation step tore-differentiate. However, those methods require relatively longperiods, around 4 weeks, for the proliferation step (Liu et al., 2007)during which the chondrocytes have to undergo several passages in orderto obtain a sufficient number. However, it has been shown thatre-differentiation is more complicated after 2 passages in particular(Hautier et al., 2008).

Others have tested the re-differentiation of chondrocytes after themonolayer proliferation step, by seeding into 3D (three-dimensional)structures known as “scaffolds”. Those structures offer the advantage ofmimicking the architecture of cartilaginous tissue. Such an environmentof is favourable to the re-differentiation of chondrocytes and to thesynthesis of extracellular matrix, as evidenced by the secretion ofproteoglycans and type II collagen (Tallheden et al., 2005). However,the prior multiplication step is deliberately shortened in order tolimit the dedifferentiation of the chondrocytes.

Seeding into a 3D structure may also offer the advantage of facilitatingin vivo implantation of chondrocytes, limiting cellular leaks andtreating large defects.

Many materials have been tested for seeding chondrocytes into a 3Dstructure; alginate, collagen, polyethylene glycol and polylactic acidmay be cited. The most appropriate are those which exhibit the followingbiological properties: cytocompatibility, low immunogenicity, andbiodegradability, with non-toxic degradation products. Thus, naturalpolymers, which have these biological properties, certainly because oftheir chemical and biological similarities with living tissue, becomethe candidates of choice. Natural polymers are highly biocompatible,readily bioresorbable and bioassimilable by the organism.

A natural polymer which is often mentioned because of its attractiveproperties as a biomaterial for three-dimensional structures ischitosan.

In fact, according to the literature, this biopolymer is known to bebiodegradable, biocompatible, non-toxic, haemocompatible, cytocompatibleand, in addition, bioactive, haemostatic, healing, bacteriostatic andfungistatic.

In addition, chitosan is known to favour cellular adhesion. It is alsoknown for its capacity to maintain the chondrocyte phenotype andstimulate the extracellular matrix synthesis process during the cultureof chondrocytes. Furthermore, no infections or allergies have beenreported which are related to chitosan. Thus, it is non-immunogenic.Regarding its resorption time, it is possible to vary it by modifyingits physicochemical properties.

Chitosan is relatively easy to use and can be produced in variousphysical forms, in particular in solutions, films (Lahiji et al., 2000),fibers, sponges, beads, hydrogels or microparticles. For this reason, ithas been used in an extremely wide variety of structures.

In the context of seeding chondrocytes into a 3D structure, a particularattention has been focused on structures that can provide both gooddistribution and maintain the chondrocytes, as well as an environmentwhich is favourable to stimulation of their chondrogenic potential. Withthis objective, various approaches have been considered. Among thevarious structures tested, beads or fragments of material, polymersintended to encapsulate the cells, and hydrogels with a pore size whichis adjusted so that the chondrocytes can be housed therein may bementioned. Some authors have envisaged injectable compositions with apolymer encapsulating the chondrocytes (WO2011/104131, University ofLiège et al). In accordance with some variations, the polymer iscross-linked in situ with the chondrocytes after injection on the defect(Hoemann et al, 2005). However, this approach suffers from thedisadvantage that the injected chondrocytes have a tendency not toremain on the injection site because of the polymerization time. Incontrast, other authors have tested some compositions, from a polymercross-linked in situ, aiming at producing a tissue which can be graftedin the form of a plug (Hao et al, 2010). However, those methods canstill cause difficulties during implantation.

In order to mimic the structure of cartilaginous tissue as best aspossible, some authors have tested the hydrogel form, and moreparticularly physical hydrogel synthesized without adding anycross-linking agent and thus favouring biocompatibility andbioresorbability of the structure.

Thus, the production of a neo-tissue has been envisaged by bringing intocontact with a physical hydrogel of chitosan (Montembault et al.).However, the prior multiplication step using monolayers is deliberatelyrapid in order to limit dedifferentiation of the chondrocytes beforeseeding, which means that multiplication of the cells is relatively low.

In a variation, the three-dimensional structure is largely resorbedduring the in vitro step for synthesis of extracellular matrix, saidstructure no longer participating in the construct (see in particularWO02078760, Laboratoires Genevrier et al). Such a method is particularlylengthy to carry out, 4 to 6 weeks, before obtaining a neo-tissue thatis capable of being implanted, and said neo-tissue is no longersupported by the 3D structure at the time of the implantation.

Another polymer which has frequently been tested in the field ofimplantation of chondrocytes is hyaluronic acid. It is a naturalpolysaccharide which is also biocompatible and biodegradable.Furthermore, it is a major component of synovial fluid andglycosaminoglycans (GAG) present in articular cartilage. Hyaluronic acidaids in protecting the joints by increasing the viscosity of thesynovial fluid and leading the cartilage more elastic. It has also beendemonstrated that hyaluronic acid favours the expression of chondrocytephenotype.

For this reason, hyaluronic acid has sometimes been combined withchitosan compositions in the culture of chondrocytes.

Some authors (Correia et al, 2011) have proposed, for example,three-dimensional sponge-like structures of a mixture of chitosan andhyaluronic acid for seeding chondrocytes. However, those structurescannot be used to obtain a homogeneous distribution of the cells; acellular gradient is observed which increases from the exterior to theinterior of the structure.

The document by Denuziere et al (1998) describes sponges ofpolyelectrolyte complexes in particular based on chitosan and hyaluronicacid. The polycationic chains of chitosan interact electrostaticallywith the polyanionic chains of the hyaluronic acid. The conclusiondrawn, however, is that sponges of chitosan alone are preferred withrespect to sponges of chitosan complexed with GAGS such as hyaluronicacid, for the proliferation of chondrocytes and for other associatedbiological properties.

A photocrosslinked hydrogel of chitosan and hyaluronic acid in whichchondrocytes are encapsulated has also been proposed (Park et al.,2013). However, encapsulation of that type does not sufficiently favourproliferation despite relatively long culture times (proliferation rate4 in 3 weeks).

Furthermore, according to the literature, although chitosan appears tohave interesting properties for the reconstruction of cartilage, itshould be noted that the degree of proliferation and synthesis ofchondrocyte extracellular matrix on sponges of chitosan alone is highlydependent on the pore size of the structure (Griffon et al, 2006).

Some authors have even considered chitosan to be unsuitable to theproliferation of chondrocytes (Suh et al, 2000).

Currently, none of these 3D (three-dimensional) environments solves theproblem of the proliferation of chondrocytes in a state compatible withthe regeneration of cartilaginous tissue with the aim to be implanted.Thus, there is a major need to obtain, by using a rapid method, asatisfactory number of chondrocytes which are differentiated and capableof generating a cartilaginous tissue with suitable properties.

Furthermore, all of the methods propose modifications in the environmentbetween the multiplication step and the differentiation step, whichinvolves losses of material and time as well as the occurrence oftrypsinization, which is damaging to the cells.

The present inventors have developed a method for obtaining chondrocytesin a sufficient number within a structure that can ensure both theirperfect multiplication, their re-differentiation and the production ofcartilaginous matrix. This structure is also suitable for implantationon the lesion.

In this context, the present inventors have found, in a completelyunexpected manner that chitosan, in the form of particles of physicalhydrogel is an excellent environment for chondrocytes, not only with aview to the synthesis of extracellular matrix of hyaline cartilage, butalso with a view to their multiplication. In fact, against allexpectations, the inventors were able to carry out, in the same chitosanstructure, a proliferation step enabling a degree of proliferation to beobtained which is similar to, or even superior to that observed usingthe ordinary monolayer technique (on plastic or in sponges) and thenreadily allowing the re-differentiation of cells. As a consequence, theinventors have developed a method that can successfully be used tomultiply chondrocytes directly after extracting in the primary state,then of inducing re-differentiation and synthesis of specificextracellular matrix in the same structure, thereby avoiding steps fortrypsinization and structure modification. In addition, this structureis compatible with in vivo implantation without necessitatingsupplemental modification.

By means of the method of the invention, it is thus possible to obtain acomposition comprising chondrocytes, distributed homogeneously withinthe structure and with a density that allows their re-implantation, in atime which is reduced compared with that which has been described untilnow, under very favourable conditions for the repair of cartilaginoustissues, in a structure directly compatible with implantation.

In the context of this description, the following terms have thefollowing particular significance: The term chitosan means apolysaccharide composed of β-(1-4)-bound D-glucosamine units(deacetylated unit) and N-acetyl-D-glucosamine units (acetylated unit).It can be produced by chemical or enzymatic deacetylation of chitin; italso exists in the natural state. In chitosan of natural origin, thepolysaccharide is generally associated with negligible quantities ofbeta-glucan. In the context of the present invention, the naturalpresence of beta-glucan in quantities in conformity with those foundnaturally in chitosan, is without influence. A chitosan is said to be“pure” even in the case of the presence of beta-glucan, as long as itspresence is below 5% by weight with respect to the polysaccharide.

The term chitosan derivative means any chitosan polymer which hasundergone a reaction aimed at modifying the chemical groups of thechitosan to change the functionalities, for example methylation,halogenation, etc. Preferably, a chitosan derivative does not have morethan three types of modification, preferably only two types ofmodification, and more preferably only one type of modification.Chitosan derivatives which are particularly considered in the context ofthe present invention are glycol-chitosan, N-succinyl chitosan, and N-or O-carboxymethyl chitosan; this list is not limiting.

The degree of acetylation (DA) is the percentage of acetylated unitswith respect to the total number of units (acetylated and deacetylatedunits). It can be determined by Fourier transform infrared spectroscopy(FTIR) or by proton NMR.

The term chitosan hydrogel means that the constituent that is in thegreat majority, i.e. more than 80% or even more than 90%, or even 95%,of the hydrogel (by weight) is chitosan, apart from water. In similarmanner, a chitosan derivative hydrogel means a hydrogel wherein theconstituent that is in the great majority, i.e. more than 80%, or evenmore than 90% or 95% of the hydrogel, is the chitosan derivative, apartfrom water.

A “hydrogel of chitosan” or “of chitosan derivative” means a hydrogelpreferably comprising at least 70% water, or even at least 80% water.

The term physical hydrogel means a hydrogel obtained by a method forgelling in an aqueous medium or in a hydroalcoholic medium not requiringthe addition of a cross-linking agent.

The term chondrocyte phenotype means cells chondrocyte-likepreferentially expressing type II collagen with respect to type Icollagen.

The term hyaluronic acid means a polysaccharide composed of D-glucuronicacid and D,N-acetylglucosamine linked together via glycoside bonds.

The term hyaluronic acid derivative means any polymer of hyaluronic acidwhich has undergone a reaction aimed at modifying the chemical groups ofthe hyaluronic acid in order to change its functionalities, for exampleby esterification.

Thus, in a first aspect, the present invention concerns an in vitromethod for obtaining a composition which is implantable by arthroscopyfor cartilaginous repair, comprising particles or fragments of aphysical hydrogel of chitosan or a chitosan derivative, and cellsforming cartilage, preferably hyaline cartilage. The compositionobtained by carrying out the method may be qualified as a cartilage gel.Such a cartilage gel in fact comprises cells synthesizing cartilaginousmatrix, all distributed homogeneously, without a cellular gradient, in athree-dimensional structure based on particles of chitosan.

Such a method in accordance with the invention in particular comprises astep for amplification of primary cells in a three-dimensional structure(3D structure), then a step for re-differentiation and induction of thesynthesis of extracellular matrix in this same three-dimensionalstructure, i.e. without changing the cells' environment.

As indicated above, chitosan is indeed a biocompatible, bioresorbablematerial which has non-toxic degradation products; it isnon-immunogenic, cytocompatible and bioactive. It is also entirelycompatible with pharmaceutical requirements as an implantable device. Inthe context of the invention, the hydrogel of chitosan or chitosanderivative is a physical hydrogel obtained without adding across-linking agent.

By means of this method, it is possible to obtain a composition orneo-tissue comprising chondrocytes within a matrix structure which isimmediately ready for implantation, with undamaged chondrocytes whichare capable of carrying out the synthesis of cartilaginous matrix afterreimplantation. Because of the absence of a change of 3D structure, themethod is easier to carry out and can be used to obtain a good qualityneo-tissue. This method is also faster than those described in the priorart.

The method in accordance with the invention is thus characterized by thefollowing steps:

-   -   (i) amplification of primary cells in a three-dimensional        structure comprising particles of physical hydrogel of pure        chitosan or chitosan derivative, then    -   (ii) induction of differentiation, or re-differentiation, and        synthesis of extracellular matrix by said amplified cells within        the three-dimensional structure of step (i).

These two steps are carried out in succession and not simultaneously,with the aim of optimizing the conditions and yields of each of thesesteps.

The Cells:

The cells which are amplified in the first step, are seeded into this 3Dstructure. They may be either chondrocytes or precursor cells ofchondrocytes obtained from stem cells, for example mesenchymal stemcells, or induced pluripotent cells (IPS). They may be any cellsdifferentiated into chondrocytes. Preferably, they are chondrocytes, andmore preferably articular chondrocytes.

In the context of the invention, it is also possible to use co-culturesof differentiated chondrocytes and/or stem cells differentiated intochondrocytes.

The chondrocytes or stem cells may be obtained using any method known tothe person skilled in the art, which can be used to recover cells from abiological sample which might contain them.

Such methods have in particular been described in the document

FR 2 965 278 (University of Caen Basse-Normandie, et al).

They are preferably human or animal cells, in particular equine orcanine cells. They may be articular cells or auricular cells, or evenobtained from the nasal septum.

Particularly preferred cells are human cells, for example humanchondrocytes, and more particularly human articular chondrocytes for ahuman patient. This is also the case for an animal, in particular ahorse or a dog.

The primary cells seeded into the structure may be allogenic, xenogenic,heterologous or autologous cells with respect to the organism which isto be treated. In accordance with a preferred implementation, they areautologous cells, i.e. they derive from the patient, human or animal tobe treated. More preferably, they are therefore autologous humanchondrocytes which could be re-implanted into the donor upon completionof the method in accordance with the invention. This is also the casefor animal chondrocytes, for example for racehorses or dogs, which couldalso benefit from a chondrocytes implantation. It may also concernxenogenic or allogenic cells, because certain measures which are wellknown to those skilled in the art could be carried out to avoidrejection during implantation.

In accordance with one embodiment, they are not human embryo stem cells.

The Three-Dimensional Structure and Chitosan:

The cells are seeded into a three-dimensional structure or scaffold, orbiomaterial, comprising fragments or particles of physical hydrogel ofpure chitosan or chitosan derivative. Said particles then form acompatible scaffold or structure which is favourable to the formation ofa three-dimensional tissue until the cells produce sufficientextracellular matrix to maintain the structure mechanically.

The cells are added after the phase for gelling and forming particles ofchitosan hydrogel. The cells are thus located on the outside of thehydrogel fragments or particles; they remain on the surface of thefragments or particles and are neither imprisoned nor encapsulated inthe hydrogel, nor do they penetrate into the pores of the hydrogel.Thus, they can move freely around the particles, as can nutrients andwaste, during the various steps of the method of the invention. Themixture produced at the beginning of the culture between the chitosanparticles and the cells mean that good distribution of the cells withinthe three-dimensional structure at the end of this method is favoured.

The chitosan used for the design of the three-dimensional structure ordevice is obtained by deacetylation of chitin, for example, which mayderive from arthropods (shrimp, insects, crab, etc), from theendoskeleton of cephalopods (squid), or from the cell wall of fungi.Depending on its origin, chitosan may be in the α conformation (cellwall of fungi, shrimp, crabs) or in the β conformation (squid) or in theφ conformation (insects), which greatly influences its biologicalproperties.

In the context of the present invention, the chitosan used may originatefrom these various sources, but a chitosan of non-animal origin willpreferably be used for reasons of biocompatibility, low endotoxinscontent, reproducibility of batches and compliance with pharmaceuticalstandards. Preferably, the chitosan used in the context of the presentinvention is chitosan extracted from the cell wall of fungi, moreparticular the common mushroom, Agaricus bisporus. In fact, in thecontext of implantable devices, because of regulatory requirements, itis particularly advantageous not to have any material which is of animalorigin. The chitosan used in the present invention, extracted fromcommon mushroom, complies with pharmaceutical requirements in terms ofthe endotoxins content, microbiological residues and heavy metals.

Succession of Steps:

The method in accordance with the invention is characterized inparticular by the succession of two steps, a first step foramplification of primary cells in a three-dimensional structure and asecond step for induction of differentiation and synthesis ofextracellular matrix (ECM) within the very same three-dimensionalstructure.

The major advantage of this method resides in the fact that it is notnecessary to change the cells' scaffold between the amplification stepand the re-differentiation step with synthesis of ECM, nor indeedbetween the re-differentiation step and that for implantation. Thismeans that it is not necessary to carry out a cell trypsinization stepat any time. In fact, after extraction, the primary cells are seededinto the structure of the invention, without a prior proliferation step,and thus without needing to detach them, in particular bytrypsinization, or by any other means that is susceptible to damage thecell wall. They then proliferate within the structure, then are inducedto re-differentiate and produce cartilaginous matrix, again without theneed to detach them, to trypsinize them, or to make them undergo anyother treatment of a nature that could damage the cell wall.

The method of the invention can thus be used to obtain cells which,after extraction, have not been damaged and have not been subjected toconditions of stress, which then can guarantee not only an optimizationof the number of cells removed, by reducing cell mortality, but alsoguarantee that at the end of the method, the cells are not in cell deathprogrammes and do not express signals which could be harmful to the hostorganism after reimplantation. At the end of one week of culture and ingeneral, throughout the culture period, the cell viability is more than90%, or even more than 93%, or even more than 97%.

Preferably, the two major steps of the method of the invention, i.e.amplification on the one hand then re-differentiation and ECM synthesison the other hand, are distinct steps. The inventors have in factobserved that the capacity to proliferate and the capacity to produceECM were preferably successive steps for cells such as chondrocytes, sothat the proliferation yields and cartilaginous matrix synthesis yieldswere much better when the two steps were distinct. In addition, thereare media which could favour one or the other of these steps,exclusively, such that it is preferable to carry them out one after theother. Preferably, the two steps as described are thus not onlysuccessive but also distinct, the second step only starting when thefirst step has been completed.

The amplification step is considered to be distinct from that forsynthesis of the cartilaginous matrix when the number of chondrocytesincreases during the first step and remains relatively stable during thesecond step with little or no amplification.

The amplification step is considered to be distinct from that forsynthesis of the cartilaginous matrix as a function of the viscosity ofthe medium: a low viscosity is observed during the amplification stepand an increased viscosity is observed during the second step,confirming the production of extracellular matrix.

Preferably, during the first step, synthesis of extracellular matrix islow.

It is also possible to observe whether such a matrix is synthesized byusing immunohistochemical methods, for example, such as those describedin the examples below.

Type II collagen (COLII) is a characteristic marker of hyalinecartilage; it is a homo-trimer with three α1(II) chains, encoded by thegene Col2a1. The analysis of this type of collagen is conventionallycarried out in order to identify differentiated chondrocytes.

Type I collagen(COLI), an α1₂ α2₁ heterotrimer produced from the genesCol1a1 and Col1a2, is conventionally considered to be a marker for thededifferentiation of chondrocytes.

The method in accordance with the invention is characterized bymaintaining the capacity of the cells which have been placed in culturewithin the structure to re-differentiate into chondrocytes. The term“maintain this capacity” means that the majority of the cells, at theend of the second step, have a chondrocyte phenotype, preferably atleast 60%, preferably at least 70%.

The inventors have in fact observed that the majority of cells seededinto the structure as described, based on hydrogel particles of chitosanor a derivative thereof, have a stable chondrocyte phenotype during thesecond step of the method for the synthesis of extracellular matrix. Atthe end of the method, the cells in the composition have no or littleexpression of type I collagen and/or do not produce it on the proteinlevel, but express a COLII/COLI differentiation index which is greaterthan 1. Preferably, the cells present in this composition synthesize theproteins of type II collagen with a COLII/COLI protein ratio greaterthan 1, preferably greater than 1.5; and/or the proportion of type IIcollagen messenger RNA is significantly higher than the proportion oftype I messenger RNA.

The method in accordance with the invention as described above canpreferably be used to obtain the cartilage composition or gel which isready for implantation in less than 40 days, preferably less than 36days, or even less than about thirty days, for example less than 28days, or indeed less than 21 days. Thus, from a biopsy of chondrocytesor of primary stem cells differentiated into chondrocytes, it ispossible to obtain a composition which is ready for implantation, havinga sufficient number of chondrocytes to be able to repair the articularlesion, in one month and a half, or less than one month, or less thanthree weeks.

To this end, the amplification step is carried out in one to threeweeks, preferably in approximately two weeks, 12 to 16 days, or even inless than two weeks.

During this step, the multiplication of the number of living cells withrespect to the number of cells initially seeded into the structure is atleast 4, or even at least 6, or even at least 7 or more than 7.

Clearly, it may be decided to prolong the multiplication step for a timesufficient to obtain a predetermined number of cells, preferablyprovided that confluence is not reached. The cell density during seedinginto the 3D structure must clearly be adapted as a consequence.

In accordance with a preferred implementation, the multiplication steplasts between 1 and 3 weeks and must be able to multiply the cellsinside the 3D structure by a ratio of at least 4, or even at least 6, oreven at least 7.

As a consequence, a biopsy of 300 mg to 500 mg of cartilage comprisingapproximately 10⁶ to 1.5×10⁶ cells can be used to obtain, at the end ofthe amplification step, 4×10⁶ cells to 10.5×10⁶ cells due to theamplification rate observed by the inventors, with this beingaccomplished in 1 to 3 weeks. Such a number of cells is considered to beappropriate for implantation of a chondrocyte construct.

The second step, linked to re-differentiation accompanying by synthesisof the extracellular matrix, may have a variable duration.

With a view to reimplantation of cartilage gel, it is, however,preferable for such a step to last between 2 and 4 weeks, preferablyapproximately three weeks, or in fact less. The duration of this secondstep may optionally be adjusted as a function of the selectedreimplantation mode.

At the end of carrying out the method of the invention, therefore, acomposition or cartilage gel is obtained comprising chondrocyte cellsdistributed in a freshly synthesized cartilaginous matrix and which arecapable of continuing the synthesis of cartilaginous tissue within a 3Dstructure composed of particles of physical hydrogel of chitosan or achitosan derivative in a manner such that said composition or cartilagegel can be directly implanted in a human being or an animal, inparticular to fill a cartilaginous articular defect, for example as aresult of traumatic and limited defects of articular cartilage, ordefects of the early superficial osteoarthritis type, or deeper defectsof the osteochondral type.

Thus, upon completion of the method, a kit or cartilage gel is obtainedwhich is ready for injection or implantation in vivo into a human beingor an animal. If the 3D structure comprising the particles of chitosanhydrogel or chitosan derivative hydrogel can be partially biodegradedduring the method, it is preferably only very slightly, preferably lessthan 50% of the initial 3D structure based on particles of chitosanhydrogel, before seeding.

Depending on the duration of the steps of the method of the invention,in particular the ECM synthesis step, the composition of the inventionor cartilage gel could also be envisaged as being injectable into ahuman being or an animal. In this respect, the duration of the secondstep for ECM synthesis will be reduced by a few days in order to ensurethat the composition remains injectable.

In contrast, in particular when it is desirable to obtain an implantwith a very specific form, the ECM synthesis step will be adjusted inorder to obtain the desired consistency to then be able to adjust theform of the composition to that which is desired. It is alsoenvisageable that the structure could be produced directly in acontainer having the desired form and of carrying out the various stepsof the method in this container in a manner such that at the end of thestep for synthesis of cartilaginous matrix, the cartilage gel has theshape induced by the container.

The composition as described above comprises hyaline cartilagesynthesized by the cells during the second step of the method, henceit's being called a cartilage gel.

A major advantage of the present invention resides in the fact that thecomposition obtained at the end of the method can be implanted directly.It is in particular not necessary to move the cells from the 3Dstructure, and thus is not necessary to make them undergo any treatment.It is also not necessary to ensure that the structure disappears, nor toawait its degradation. In contrast, in accordance with the method of theinvention, the chitosan based structure is still present at the end ofthe ECM synthesis step and forms part of the composition or neo-tissueintended to be implanted. In fact, such a structure acts as a scaffoldto maintain the cells in a suitable environment so that, afterimplantation, it enables ECM to carry on its synthesis, and thus to beperfectly filled into the lesion, for example an articular lesion. Thechitosan structure thus does not act solely to favour the synthesis ofECM in vitro, but also, after reimplantation, to support the implantedcells; thus, it participates in the structure of the reimplantedneo-tissue.

The structure also ensures an optimized spatial distribution of seededcells, and thus allows the synthesized ECM to be distributedharmoniously during the method of the invention and also afterreimplantation.

The number of cells implanted in the structure of the invention can varyas a function of the size of the lesion which it is to be filled, andalso as a function of the number of cells which it is planned to collectfor the purposes of seeding. However, preferably, at least approximately10⁵ primary cells are seeded, in particular at least approximately 6×10⁵primary cells, or at least 10⁶ primary cells, preferably human, canineor equine primary chondrocytes; at the end of the method of theinvention, the final composition preferably comprises at least 3×10⁵chondrocytes, or at least 6×10⁶ chondrocytes, or perhaps more. In orderto obtain a composition which can be implanted directly into acartilaginous lesion, 10⁶ to 1.5×10⁶ cells are preferably seeded inorder to obtain 4 to 10.5×10⁶ cells upon completion of the amplificationstep.

In accordance with one implementation of the invention, the chondrocytesin the composition have a concentration of approximately at least 10⁶cells/g, preferably approximately at least 6×10⁶ cells/g of 3D structureat the time of the beginning of the culture.

Culture Media:

In accordance with a particularly preferred embodiment, two differentculture media are used. One is for the first amplification step and theother is for the second step for induction of re-differentiation andsynthesis of ECM; the passage from one to the other of the steps istherefore carried out by modifying the medium, the two media beingdistinct.

In particular, during the first step, a medium favouring theproliferation of cells is used without inducing ECM synthesis. Theproliferation of cells is generally accompanied by a phenomenon ofdedifferentiation; however, a medium is used which preserves theircapacity to re-differentiate into chondrocytes at the end of theproliferation step.

The inventors have in fact evidenced that within a three-dimensionalstructure, it is preferable to carry out an intense amplification stepwithout in any way, inducing the synthesis of ECM.

Indeed, the three-dimensional structures and chitosan were known beforethe invention for their capacity to favour the chondrocyte phenotype bylimiting dedifferentiation during the extracellular matrix synthesisstep. Completely unexpectedly, the inventors have shown that it waspossible to amplify the chondrocytes without inducing massive synthesisof extracellular matrix in a three-dimensional structure based on achitosan hydrogel or a derivative.

A particularly suitable medium for the proliferation step is a mediuminducing amplification, in particular a medium comprising fibroblastgrowth factor (FGF-2), and also insulin, corresponding to the mediumtermed “FI”, as illustrated in the experimental section (Claus et al.,2012). FGF-2 is preferably present in a concentration between 2 and 10ng/mL, and insulin is present in a concentration between 2 and 10 μg/mL.However, other culture media which are well known to the person skilledin the art may also be used, in particular any of the culture mediamostly used for this kind of cell, but in monolayers. Because of thethree-dimensional structure and the chitosan, the cells seeded insidethe structure of the invention mainly conserve their round morphologyand their capacity to subsequently re-differentiate into chondrocytes.

For the culture medium of the second step, media to be employed arethose conventionally used in order to favour the differentiation orre-differentiation of dedifferentiated chondrocytes and to allow thespecific synthesis of ECM. Such media are well known to the personskilled in the art. A particularly preferred medium comprises BMP-2(bone morphogenetic protein 2); preferably, the medium is that used inthe experimental section, in particular composed of BMP-2 and insulin,and preferably also triiodothyronine T3 (Liu et al., 2007, Claus et al.,2012), corresponding to a medium called “BIT”. The BMP-2 is preferablyin a concentration in the range 100 to 500 ng/mL, with the insulin in aconcentration between 2 and 10 μg/mL and the triiodothyronine, T3, in aconcentration between 50 and 250 mM.

The BMP-2 will preferably be from the same species as the cells used,i.e. a human BMP-2 for human cells. This is also the case for animalcells.

As was the case for the culture medium of the first step, culture mediaare preferred for the second step, which do not oppose subsequentreimplantation of the composition at the end of the ECM synthesis step.This in particular prevents the compounds from running the risk ofgenerating rejection reactions which are incompatible with theRegulation for implantable devices.

In addition, steps for eliminating the culture medium before injectionor reimplantation at the end of the two steps mentioned above may beenvisaged.

The first and the second step may independently be carried out undernormoxic or hypoxic conditions.

Production of Chitosan Hydrogel:

The hydrogel of pure chitosan or chitosan derivative is produced fromchitosan, preferably extracted from fungi and having a weight averagemolecular weight (Mw) which is preferably more than 150 kDa (i.e. 150000 g/mol) in order to favour the physical gelling process by thepresence of long macromolecular chains. It is preferably in the range150 to 220 kDa.

If the chitosan has a substantially different molar mass, in particularif it is extracted from another source, the method for producing thephysical hydrogel of chitosan, as described in the experimental section,could readily be adapted by the person skilled in the art usingtechniques which are well known.

In addition, it is also possible to use chitosan for the hydrogel whichhas a variable degree of acetylation; preferably, however, theacetylation degree of the chitosan is in the range 5% to 60%, preferablymore than 25%, for example between 25% and 60%, or between 28% and 40%.This acetylation degree in effect induces an environment which isfavourable to cells, leading to good adhesion between the hydrogelformed and the cells, and to good chondrogenesis results.

With a view to the formation of hydrogel, a solution of chitosan ispreferably used wherein the concentration is sufficiently high to allowthe macro molecular chains to become entangled and thus to favourphysical gelling. In the context of the invention, the hydrogel is infact obtained by an entirely physical process without using any chemicalcross-linking agent. The concentration of chitosan in solution may be inthe range 0.5-4% (w/w), preferably more than 1.5%, or even 2%.Preferably, the chitosan or the chitosan derivative has a concentrationby weight in the hydrogel in the range 3.4% to 4.2%, beforeneutralization.

Several chitosan gelling methods may be used in the context of thepresent invention. The following particularly suitable methods may inparticular be used, such as physical gelling with gas (ammonia) orphysical gelling in a hydroalcoholic or aqueous medium.

Preferably, the hydrogel used in the context of the present invention isobtained by means of an evaporation method carried out in an alcoholicmedium, as illustrated in Example 1 in the experimental section; such amethod is also known as hydroalcoholic gelling.

The hydrogel obtained preferably has a thickness between 3 and 5 mm.

The pore size of the hydrogel obtained must be both smaller than thesize of the cells and also sufficient to allow free diffusion ofnutrients and elimination of waste. The embodiments described above andimplemented in the experimental section can be used to obtain a poresize of this type. It should be noted that in the context of the presentinvention, the hydrogel of chitosan or chitosan derivative is such thatthe pore size cannot allow the penetration of cells into the interior ofthe hydrogel. The cells seeded into the three-dimensional structure inaccordance with the invention thus proliferate without penetratinginside the hydrogel. Because the cells do not multiply in the pores ofthe hydrogel, and they can move freely around the hydrogel particles,this means that a homogeneous distribution of the cells inside thestructure can be obtained without the substantial cellular gradientsreported in Correia et al., 2011.

The person skilled in the art will be able to determine the pore size ofa hydrogel and adjust the parameters for its production in order toensure that the pore size is sufficiently small to prevent cells frompenetrating, in particular chondrocytes, while nevertheless allowing thefree diffusion of nutrients.

In order to obtain hydrogel particles constituting the base of thethree-dimensional structure of the invention, the hydrogel obtained inthis manner is manipulated using any suitable means well known to theperson skilled in the art; this ensures that the hydrogel is fragmentedinto particles.

The hydrogel particles obtained in this manner are irregular in shape,but preferably have a relatively homogeneous size distribution, i.e. 50%of the particles have a size between −20% and +20% of the mean size. Inaccordance with a preferred implementation, the particles have a meansize in the range 10 μm to 1500 μm; preferably between 200 μm and 1200μm (1.2 mm), and more preferably between 400 μm and 700 μm. The term“particle size” means the length of the edge if the particles areassimilated to rectangles, the length of the largest diameter if theparticles are assimilated to ellipses. The particles are preferablyformed as ellipses.

The inventors have demonstrated the best results for particles with amean size over one hundred microns, in particular above 60 μm, or infact above 400 μm and less than 1.2 mm.

A certain variability in the particle sizes of the chitosan hydrogelparticles appears to be favourable to the culture of chondrocytes.

In accordance with a preferred embodiment, the hydrogel is initiallyobtained by gelling using the hydroalcoholic route, then fragmentedusing any appropriate means.

Design of the Three-Dimensional Structure:

The three-dimensional structure in accordance with the invention thuscomprises particles of physical hydrogel of chitosan or one of itsderivatives, as detailed above, constituting a three-dimensionalstructure within which the cells are seeded or migrate naturally. Inaccordance with a preferred embodiment, the cells are mixed with theparticles of hydrogel. The physical hydrogel of pure chitosan or ofchitosan derivative is preferably solely constituted by chitosan or aderivative and water in an amount which is preferably at least 70%,preferably at least 80%. In particular, this hydrogel compositioncontains neither chemical cross-linking agent nor any other polymer, inparticular polysaccharide or a derivative, apart from the β-glucannaturally associated with chitosan. The large percentage of waterensures that it is in fact a hydrogel of chitosan and not a sponge-likestructure or other structure obtained by lyophilization of a chitosansolution. It should be noted that the large percentage of water in thehydrogel can mimic the natural environment of chondrocytes as best aspossible, since the cartilaginous tissue comprises approximately 80%water.

In accordance with a particularly preferred embodiment, an anionicmolecule is added to the hydrogel particles of chitosan or chitosanderivative in order to reinforce the mechanical and biologicalproperties of the three-dimensional structure. This molecule does notform a part of the composition of the hydrogel, but is a constituentadded after gelling the hydrogel of chitosan or chitosan derivative, andpreferably after producing the hydrogel particles. The anionic moleculethus interacts only at the surface of the hydrogel particles inaccordance with the invention, thereby favouring the formation of“hairy” chitosan particles by linear chains of this anionic molecule.

The anionic molecule associated with the surface of the chitosanparticles is preferably in the form of a polymer, for example hyaluronicacid, or chondroitin sulphate. The chitosan chains have positive chargesdue to the amine groups in the protonated form, NH₃ ⁺, and the chains ofthis anionic molecule interact via electrostatic bonds, thereby forminga stable complex in physiological medium (pH in the range 5 to 8, andespecially in the range 6 to 7). The anionic molecule can thus be usedto electrostatically cross-link the fragments or particles of hydrogelby interacting with the cationic chains of chitosan on the periphery ofthe particles, thereby reinforcing the mechanical properties of thethree-dimensional structure or scaffold within which the cells areseeded.

It should be noted that the anionic molecule may be added to thethree-dimensional structure before seeding with primary cells, and thusbe present during the step for proliferation and ECM synthesis, and thusalso during the subsequent implantation of the composition. In thecontext of the invention, seeding primary cells into a three-dimensionalstructure free from the anionic molecule and adding the latter againeither during the first multiplication step or at the end of the firststep when the second re-differentiation step is begun, or at the end ofthe second step for re-differentiation and ECM synthesis beforeimplantation, may also be envisaged.

The anionic molecule will preferably be hyaluronic acid, one of thecomponents of synovial fluid, known for its chondroprotective propertiesand favourable to chondrogenesis. Thus, the quantity of hyaluronic acidnecessary to modify the viscoelastic characteristics of thethree-dimensional structure is added. The electrostatic interactionsoccur between the amine groups in the protonated form, NH₃ ⁺, ofchitosan and the carboxyl groups of the hyaluronic acid.

A derivative of hyaluronic acid or a complex of hyaluronic acid may alsobe used. The relative proportion of the anionic molecule with respect tothe chitosan hydrogel is preferably in the range 1% to 10%, preferablyin the range 1% to 3%.

The hyaluronic acid may be from animal origin, for example by extractionfrom rooster comb, or from non-animal origin, obtained by bacterialfermentation. In the context of the present invention, the hyaluronicacid will most preferably be selected to be from bacterial origin. Infact, the hyaluronic acid obtained by bacterial fermentation is knownfor its better properties of biocompatibility, thereby avoidingallergies and rejection, reproducibility of batches and compliance withpharmaceutical standards. The hyaluronic acid used in the context of thepresent invention complies with pharmaceutical standards.

Its molecular mass by weight is preferably 50 kDa to 4 MDa, and ispreferably selected to be more than 500 kDa, preferably between 500 kDaand 2 MDa, for example between 1 MDa and 2 MDa.

More particularly, preferably in the context of the present invention,the components of the three-dimensional structure, which are hydrogelparticles of chitosan or chitosan derivatives, with or without theaddition of chains of anionic compound, must be resorbable in vivo. Inorder to obtain such a property, it is important that none of thecomponents of the three-dimensional structure oppose its resorbablenature.

Preferably, the association of hydrogel particles of chitosan orchitosan derivatives, with or without the chains of anionic compound,will be resorbed after several weeks once implanted, for example afterat least two weeks, preferably at least 4 weeks. It is generallypreferable for the resorption time not to exceed 6 months, preferablynot to exceed 4 months. Depending on the type of application envisaged,the resorption time may be adjusted by the person skilled in the art.

It is important to note that the compositions in accordance with thepresent invention may be adapted in terms of form, diameter,concentration, and content depending on the various targetedapplications. In particular, the three-dimensional structure formed ofhydrogel particles of chitosan or chitosan derivative with or withoutthe chains of anionic compound may be produced in a manner such that itsshape corresponds to that of the lesion observed into which it will bereimplanted upon completion of the method in accordance with theinvention.

However, no dehydration step is necessary nor desired prior toreimplantation.

As discussed above, the method may be used to obtain a cartilage gel orcomposition which is ready for implantation or injection in vivo, inparticular for a human being or an animal such as a dog or a horse.

However, prior to implantation, the medium may be changed, or additionalcompounds may be added, in particular compounds which are soluble in thethree-dimensional structure. As an example, adding pharmaceuticalcompounds such as anti-inflammatory agents, anaesthetics, analgesics,corticosteroids, vitamins, minerals, compounds aiming at reducing theimmune response and/or compounds favouring grafting may be envisaged,including all or a part of these compounds or a combination of thesecompounds; this list is not limiting.

In accordance with a second aspect, the present invention concerns athree-dimensional structure formed by particles of physical hydrogel ofchitosan or a derivative of chitosan and an anionic molecule associatedwith these particles. Such a matrix may advantageously be used to seedchondrocytes, causing them to proliferate then synthesize extracellularmatrix, more particularly matrix characteristic of hyaline cartilage, inparticular for use in accordance with the present invention, beforebeing implanted or injected in the form of cartilage gel, in particularinto an articular defect. As demonstrated in the context of the presentinvention, such a structure can in fact be used to ensure not only theproliferation of cells such as chondrocytes, but also to provide anenvironment which is particularly favourable to the synthesis of hyalinecartilage matrix. The invention also concerns an implantable orinjectable composition comprising this three-dimensional structure anddifferentiated chondrocytes that are capable of synthesizingcartilaginous tissue. It should be noted that the composition orcartilage gel also comprises cartilaginous matrix synthesized by thechondrocytes contained in the composition.

The various elements mentioned with regard to the method of theinvention, are as described for the first aspect of the invention, inparticular the three-dimensional structure, the chitosan or itsderivative, the hydrogel, the particles and the anionic molecule. Thepreferred implementations in the context of this first aspect are alsopreferred in the context of this second aspect. In particular, thechitosan is preferably a chitosan obtained from fungi, and moreparticularly extracted from the cell wall of the common mushroom,Agaricus bisporus. The particles have the specific sizes given above,namely between 10 μm and 1200 μm, preferably between 400 and 700 μm onaverage. Thus, the structure is preferably a 3D structure formed byparticles of physical hydrogel of chitosan with a mean size in the range400 μm to 700 μm, where said chitosan is extracted from the commonmushroom.

Concerning the anionic molecule, as explained for the method of theinvention, this is preferably a polymeric molecule and more preferablyhyaluronic acid or a derivative of hyaluronic acid or a complex ofhyaluronic acid, and more specifically hyaluronic acid derived frombacterial fermentation.

The differentiated chondrocytes present in the composition of theinvention are articular chondrocytes, for example. More preferably, theyare human or animal chondrocytes, in particular canine or equine. Thechondrocytes are differentiated chondrocytes with a chondrocytephenotype, in particular with a COLII/COLI differentiation index greaterthan 1. Preferably, the chondrocytes present inside this compositionprincipally synthesize proteins of type II collagen with a COLII/COLIprotein ratio greater than 1, preferably greater than 1.5; and/or themessenger RNA content of the type II collagen is significantly higherthan the messenger RNA content of type I collagen, with a COLII/COLItranscriptional ratio for the cells higher than 1, for example more than100, or more than 1000.

In accordance with one implementation of the invention, the relativeproportion of chondrocytes inside the composition corresponds to aconcentration in the range 10⁶ and 10⁷ cells/g of 3D hydrogel structure,when culture is begun.

The composition of the invention may also comprise other compounds ormolecules and in particular extracellular matrix (ECM).

Preferably, a composition as described or cartilage gel is capable ofbeing obtained by carrying out the method of the invention, inparticular by culturing cells inside the three-dimensional structure,then proliferating them, followed by their re-differentiationaccompanied by ECM synthesis.

It is also possible to envisage adding additional compounds, inparticular soluble compounds, for example selected fromanti-inflammatory agents, anaesthetics, analgesics, corticosteroids,vitamins, minerals, compounds aimed at reducing the immune responseand/or at favouring grafting, all or a portion of these compounds or acombination of these compounds; this list is not limiting.

As described in the context of the method of the invention, thethree-dimensional structure as described is advantageously resorbable,in particular bioresorbable in vivo. The properties of the hydrogel ofchitosan or a derivative of chitosan will be selected as a function ofthe timescale desired for integral resorption of the three-dimensionalstructure, once the composition has been implanted. Preferably, theresorption time will be adapted such that such a resorption occursconcomitantly with the synthesis of the cartilaginous matrix by thechondrocytes present in the composition; preferably, the resorption timewill be adjusted in a manner such that the cartilaginous matrix formedby the chondrocytes substitutes itself in its entirety into thethree-dimensional structure of the hydrogel of chitosan or chitosanderivative.

In accordance with another aspect of the invention, the composition asobtained at the end of the method of the invention or as described inaccordance with the second aspect of the invention is for therapeuticand/or surgical use, in particular for use as an implant or construct inthe repair or reconstruction of cartilaginous tissue, or in thetreatment of osteoarthritis, and more generally in the treatment of anydisease characterized by a degradation or disappearance of cartilaginoustissue, in particular cartilaginous defect, for example followingtraumatic and limited defects of articular cartilage, or deeper defectsof the osteochondral type. Such a composition for in vivo use isenvisaged in particular for surgery, for rheumatology or as a vector foran active principle. The composition or cartilage gel can be implantedby arthroscopy.

A particular envisaged use is in chondrocytes implantation. Preferably,the chondrocytes present inside the composition to be implanted areautologous or allogenic cells, preferably human, canine or equinechondrocytes.

In accordance with a further aspect, the present invention also concernsa three-dimensional structure formed by particles or fragments ofphysical hydrogel of pure chitosan or a derivative of chitosan and itsuse for seeding cells in vitro, in particular for the purposes ofproliferation and synthesis of extracellular matrix, in particular forcarrying out the method in accordance with the first aspect of thepresent invention. The three-dimensional structure is that describedabove; this is also the case for the hydrogel of chitosan or aderivative of chitosan. This hydrogel is preferably extracted from thecommon mushroom as explained above, with a weight average molecularweight which is preferably in the range 150 to 220 kDa. The hydrogelparticles preferably have a mean size in the range 200 μm to 1.2 mm,more preferably in the range 400 to 700 μm. An anionic molecule ispreferably added to the hydrogel particles of the three-dimensionalstructure; it is preferably an anionic polymer, more particularlyhyaluronic acid or a derivative of hyaluronic acid or a complex ofhyaluronic acid, in particular obtained by bacterial fermentation.

All of the preferred implementations detailed above concerning theelements of the three-dimensional structure are also applicable to thisaspect of the invention, and more particularly to the followingcharacteristics: the hydrogel particles preferably have a size in therange 200 μm to 1.2 mm, preferably in the range 400 to 700 μm, and/orthe chitosan has a weight average molecular weight which is preferablymore than 50 kDa, preferably in the range 150 to 220 kDa, and/or thechitosan has a degree of acetylation in the range 5% to 60%, preferablyin the range 28% to 40%, and/or the hyaluronic acid has a weight averagemolecular weight in the range 50 kDa to 4 MDa, preferably in the range 1to 2 MDa.

The proportion of hyaluronic acid with respect to the chitosan hydrogelis preferably in the range 1% to 10%, preferably in the range 1% to 3%.As described above, this three-dimensional structure is advantageouslyused for seeding or culturing primary cells, in particular primarychondrocytes, or primary stem cells differentiated into chondrocytes, inparticular mesenchymal stem cells. However, other kinds of cells may becultured in this three-dimensional structure, in particular bone cells,fibroblasts, keratinocytes, or combinations of certain of these cells;this list is not limiting. The present inventors have in factdemonstrated that this three-dimensional structure provides athree-dimensional architecture which is particularly favourable tocells, whether they are in proliferation or multiplication phases or inphases for the synthesis of extracellular matrix. Further, as describedabove, this three-dimensional structure is biodegradable andbioresorbable, and may therefore be implanted in vivo, into humans oranimals, once seeded by the cells.

In the context of the present invention, in vivo implantation of athree-dimensional structure in accordance with the invention, i.e. athree-dimensional structure comprising fragments or particles ofphysical hydrogel of chitosan or a chitosan derivative,electrostatically cross-linked by an anionic molecule, preferably apolymer, in particular hyaluronic acid or a hyaluronic acid derivativeor a hyaluronic acid complex, said structure being free from cells suchthat the 3D structure is precisely colonized in vivo by the cells, mayalso be envisaged.

The three-dimensional structure as described in the context of thevarious aspects of this invention is preferably sterilized before beingseeded.

FIGURES

The patent or application file contains at least one color drawing.Copies of this patent or patent application publication with colordrawing will be provided by the USPTO upon request and payment of thenecessary fee.

FIG. 1 : scanning electron microscopy micrograph of a physical hydrogelof pure chitosan.

FIG. 2 : scanning electron microscopy micrograph of a physical hydrogelof pure dehydrated chitosan

FIG. 3 : optical microscopy image of a physical hydrogel of purechitosan after treatment with eosin.

FIG. 4 : shows the viability rates for cells seeded into a M1-type 3Dstructure as a function of the initial density of chondrocytes, measuredwith the Live and Dead kit on 7-day culture fractions (duringamplification, in FI medium). The count of the dead cells (in black) andlive cells (grey) was carried out with ImageJ software from fluorescencemicroscopic images with a magnification of ×20. The percentage of deadcells was calculated for each condition by calculating the deadcell/total cell ratio.

FIG. 5 : shows the evolution as a function of time of the population ofchondrocytes in 3D structures based on particles of physical hydrogel ofchitosan extracted from fungi supplemented or not with hyaluronic acid,with different particle sizes or in 3D structures based on particles ofphysical hydrogel of chitosan extracted from squid, or also ofchondrocytes cultured in monolayers under “FI” culture conditions. Thenumber of cells is along the ordinate; the culture time in days is alongthe abscissa.

FIG. 6 : shows the evolution in the population of chondrocytes as afunction of time for different initial densities of chondrocytescultured as monolayers, under “FI” culture conditions, confirming thatthe cell population obtained is identical beyond 7 days irrespective ofthe initial density.

FIG. 7 : optical microscopic images as a function of time ofchondrocytes cultured inside the three-dimensional structures of purehydrogel particles (M1).

FIG. 7A: represents cells obtained from the amplification step in FImedium 14 days after seeding.

FIG. 7B: represents cells during the ECM synthesis step in BIT medium 24days after seeding, i.e. 10 days after inducing re-differentiation andECM synthesis. The magnification ratio is ×20.

FIG. 8 : shows the quantity of messenger RNA of type I collagen and typeII collagen relative to the GAPDH gene, measured by quantitative RT-PCRfor chondrocytes cultured in a 3D structure (M1) with several initialcell densities compared with the monolayer technique, after 35 days ofculture.

FIG. 9 : shows the ratio of messenger RNA for the COLII/COLI genesobtained by quantitative RT-PCR, for chondrocytes seeded in a 3Dstructure (M1) with several initial cell densities compared with themonolayer technique, after 35 days of culture.

FIG. 10 : shows Western blot analysis of the protein counts for type Iand type II collagen, for chondrocytes cultured in a three-dimensionalstructure M1 compared with chondrocytes cultured in monolayers, after 35days. The level of expression of actin acts as a control.

FIG. 11 : shows the analysis, by immunohistochemistry, for chondrocytescultured in 3D structure M1 and M2 compared with those cultured inmonolayers (×20) (MC) after 35 days, for the same initial cell density(6×10⁵ cells) by HES and SO staining, as well as immunolabelling of typeI collagen and of type II collagen.

EXAMPLES Example 1: Synthesis of Physical Chitosan-Based Hydrogel

The chitosan used was from non-animal origin, extracted from the cellwall of the common mushroom, Agaricus bisporus. Its weight averagemolecular weight (Mw) was 170 g/mol; and its degree of acetylation (DA)was 32%. It was used in the form of a powder.

The pure chitosan was dissolved in an acidic solution of acetic acid (1%in water), in stoichiometric amounts with the amine groups of thechitosan. The solution was stirred at room temperature until thechitosan had completely dissolved, i.e. for at least 3 h, preferably atleast 6 h.

Next, 1,2 propanediol was added in a quantity identical to that of theacetic acid and stirring was continued for at least 30 min, preferably 1h at room temperature. The mixture could then be degassed at roomtemperature, or under vacuum if necessary if the solution shows a lot ofair bubbles.

The solution was then poured into containers like multi-well plates or 3cm petri dishes, then it was left to stand, preferably overnight. Thesolution was then placed in a vacuum oven, preferably at 50° C., for thetime necessary to allow a gel to form, preferably at least 20 hours.

The gelling step could also be carried out at room temperature, but thenwould have required longer times (5-8 days depending on the intrinsiccharacteristics of the chitosan).

The thickness of the solution before gelling could be in the range 2 to7 mm, preferably in the range 3 to 6 mm, in order to favour evaporationand hydrophobic-like interactions for good gel setting.

The physical hydrogel obtained was then neutralized in a basic mediumwith a 0.1N sodium hydroxide solution, preferably for 1 h. Next, severalwashes with water were carried out, preferably with sterile water. Eachwash preferably lasted approximately 1 hour in order to remove excessalcohol and bring the hydrogel to a neutral pH. In general, at least 6washes were carried out.

The gel obtained thereby had a water content of approximately 80% byweight. The final concentration by weight of chitosan in the hydrogelwas in the range 1% to 4.5% before neutralization, preferably between3.4% and 4.2% before neutralization.

It is important to control the temperature and humidity conditionsduring the synthesis of the chitosan-based hydrogels, more particularlywhen it is extracted from fungi, preferably under room temperatureconditions which are below 25° C.

The hydrogel obtained at the end of these various steps had a thicknessof 3 to 6 mm, preferably between 4 and 5 mm thick, and was a translucentwhite colour and its surfaces were smooth and regular. However, itsappearance could vary as a function of the intrinsic properties of thebasic chitosan, in particular the degree of acetylation, the molar massand the concentration. It was in the form of a viscoelastic block withmechanical properties which depended on the intrinsic characteristics ofthe starting chitosan, in particular and once again the degree ofacetylation, the molar mass and the concentration.

The hydrogel obtained was easy to manipulate, detached withoutdifficulty and without tearing the flat surface on which it had beenproduced.

Conventional scanning electron microscopic observation of the dehydratedhydrogel showed a fibrillar 3D structure, porous, similar to that of aliving tissue, as can be seen in FIG. 2 . Scanning electroncryomicroscopic observation of the hydrated hydrogel, as can be seen inFIG. 1 , showed a pore size between 1-3 μm, which did not allow cells topenetrate inside the hydrogel but allowed free diffusion of nutrientsand cellular waste.

Example 2: Synthesis of Particles/Fragments of Chitosan Hydrogel inOrder to Produce the 3D Structure (Structure M1 and Structure M2)

Structure M1:

The chitosan hydrogel obtained at the end of Example 1 was cut intosmall squares with 1 mm sides then placed in 10 mL of water, preferablysterile water. The hydrogel was then ground with the aid of an UltraTurrax, at 6000 to 17000 rpm for 10 seconds, carrying this out 2-4times. In order to obtain particles with a homogeneous size and theexpected diameter, grinding was preferably carried out at 6000 rpm for10 seconds repeated 3 times, in order to obtain particle sizes of:400-700 μm (50%), or in fact 250-900 μm (>80%), with a mean of the orderof 650 microns. The solution obtained was centrifuged, preferably at1375 g for 7 minutes, in order to recover the pellet constituted byparticles of chitosan hydrogel. FIG. 3 illustrates an example of thechitosan particles obtained. A mini-spoon was used to measure thequantity of particles of chitosan which would be brought into contactwith the chondrogenic cells. Preliminary tests validated thereproducibility of the measurement.

Structure M2:

In order to reinforce the viscoelastic properties of the 3D structure inwhich the chondrocytes were seeded, the inventors also produced a secondstructure (M2) by adding an anionic constituent, interacting with thecationic functions of the chitosan. The selected polymer was hyaluronicacid, preferably from bacterial origin, since such a constituent isknown for its better biocompatibility properties, in order to avoidallergies or any rejections. The molecular mass by weight of thehyaluronic acid used in producing the structure M2 was approximately 2MDa. The hyaluronic acid was added after preparing the chitosan hydrogelparticles.

Example 3: Culture of Cells in 3D Structure

The cells used in the context of this example were human chondrocytesobtained from human samples and treated in accordance with the protocoldescribed in the document FR 2 965 278 (University of CaenBasse-Normandie, et al).

The hydrogel particles obtained at the end of Example 2, with hyaluronicacid (3D structure M2) or without hyaluronic acid (3D structure M1) weresterilized, for example at 121° C. for 15 minutes, prior being broughtinto contact with the cells. Several mini-spoons of hydrogel particleswere removed, preferably 2 mini-spoons corresponding to 80 to 84particles, which were introduced into the wells of a 24-well cultureplate which had been covered with an insert (pore size 8 μm). The cellswere added thereto, between 10⁵ and 10⁷, preferably of the order of6×10⁵ cells/wells, per 80-84 particles of hydrogel, which were mixedcarefully with the chitosan hydrogel particles. This proportion of cellswith respect to the 3D structure corresponded to approximately 6.7×10⁶cells per gram of 3D structure at the moment of seeding.

Culture was carried out in a controlled atmosphere in an oven at 37° C.,with a CO₂ percentage of 5% under normoxic conditions.

The cells adhered spontaneously to the chitosan hydrogel particles. Thequantity of cells falling to the bottom of the well was considered to benegligible.

As a control, 6×10⁵ cells obtained as described above were cultured inmonolayers on plastic in 24-well plates under culture conditions whichwere identical to those described above for the 3D structures(controlled atmosphere in an oven at 37° C., with a CO₂ percentage of5%, normoxia). The cells also adhered there spontaneously.

Example 4: Cell Proliferation Step

In this step, the selected culture medium was favourable to themultiplication of cells.

The selected medium was a 50/50 solution of DMEM-HAM F12+1% AB(streptomycin/penicillin)+10 FCS supplemented with “FI” solutioncomprising FGF-2 in a concentration of 5 ng/mL+insulin in aconcentration of 5 μg/mL. (Claus et al; 2012).

After a short period, the mixture described in the preceding step wasrecovered from this culture medium which is known to favour theproliferation of cells.

During this amplification phase, the culture medium was renewed 3 timesper week for the cultures in the 3D structures, as was the case for themonolayer cultures. The proliferation period lasted between one and twoweeks in order to obtain a sufficient number of cells.

The inventors observed that the amplification phase lasted approximatelytwo weeks when the cells were seeded into a structure constituted byparticles of pure hydrogel (3D structure M1) and could be shortened to 1week in the presence of hydrogel particles supplemented by linear chainsof an anionic molecule such as hyaluronic acid (3D structure M2).

Furthermore, the initial quantity of 6×10⁵ cells/insert could of coursebe increased provided that the condition regarding number of cells/massof hydrogel or number of cells/volume of hydrogel or number ofcells/number of hydrogel particles is adhered to. By way of example, itis entirely possible to seed 1 to 1.5×10⁶ cells/insert, provided thatthe necessary quantity of 3D structure is added in order to obtain morethan 4×10⁶ cells/insert at the end of the method, or even 10.5×10⁶cells/insert, or more.

Analysis by Optical Microscopy:

Analysis by phase contrast microscopy was carried out. It confirmed thatthe cells adhered well to the particles of chitosan hydrogel and thatthis environment was favourable to their culture. The culture conditions(three-dimensional structure M1 or M2, and FI culture medium) favouredthe proliferation and division of the chondrocytes.

The cells observed could proliferate either in an isolated manner or inclusters/bunches. The cultures carried out in 3D structures of hydrogelparticles exhibited mainly round cells. The elongated form,characteristic of fibroblasts, was not observed in the 3D structures M1and M2, except occasionally at the periphery, i.e. at the interfacebetween the structure and the external medium.

As a control, the inventors carried out monolayer cultures at the sametime. After 24 hours of culture, in the same FI medium as the cellsseeded into the structures M1 and M2, the chondrocytes adopted anelongated morphology characteristic of fibroblastic cells.

Viability of Cells:

The viability of the chondrocytes seeded inside the three-dimensionalstructures was measured with the Live and Dead kit on fractions ofcultures at 7 days (during amplification, in FI medium). The dead cells(red) and live cells (green) were counted using ImageJ software fromfluorescence microscope images, magnification ×20. The percentage ofdead cells was estimated for each condition by calculating the deadcell/total cell ratio. The viability was more than 93%, or in fact morethan 97%, which demonstrated good compatibility with the 3D structure.

FIG. 4 illustrates the obtained results.

Proliferation Tests:

Proliferation tests were carried out by counting the total cells usingthe Cellometer T4 after detaching the cells with trypsin and stainingthe dead cells with trypan blue.

The measurements were carried out after 1 day (D1), 14 days (D14) and 21days (D21) of culture after seeding the primary chondrocytes at D0.

FIG. 5 illustrates the evolution in the cellular population.

After 7 days, the increase in the number of cells was clearly observed.The cells survived and proliferated very well in the 3D structure aswell as in monolayers (MC).

In the three-dimensional structures, M1 or M2, the cells remained roundduring the multiplication step, while they adopted an elongated shapelike fibroblasts in monolayers.

Further, FIG. 6 illustrates that in monolayers, the cell population wasidentical from 7 days, irrespective of the initial density of the seededcells.

In conclusion, at the end of this proliferation step, amplification ofthe cells in a three-dimensional structure composed of particles of purechitosan hydrogel (structure M1) were observed to be almost asproductive as in monolayers (MC), which is the reference protocol forthe multiplication of cells such as chondrocytes, but it does involve atrypsinization step which can be avoided by using the structure M1.

Adding hyaluronic acid to the three-dimensional structure of chitosanhydrogel particles (structure M2) induced a very strong acceleration ofcell proliferation, much greater than the M1 structure or the monolayerculture, in particular by a ratio of two.

Example 5: Differentiation and Production of Extracellular Matrix

In the context of the present invention, the steps for multiplicationand differentiation were preferably distinct: firstly, the cells aremultiplied and secondly, they are differentiated and produceextracellular matrix. The culture medium used for the precedingmultiplication step was modified after 15 days. The culture medium “FI”was replaced by a medium “BIT” with the aim to favouring the step fordifferentiation of cells and the production of extracellular matrix.

Thus, the culture medium was preferably composed of: 50/50 DMEM-HAMF12+1% AB (streptomycin/penicillin)+10 FCS, to which a BIT solutioncomposed of the following was added: BMP-2, 200 ng/mL+insulin, 5μg/mL+triiodothyronine, T3, 100 mM (Claus et al, 2012).

This fresh culture medium was renewed every two or three days. Theperiod for re-differentiation and chondrogenesis preferably lasted 3weeks. As a control, the same medium change was employed as for thecells cultured in monolayers.

Analysis by Optical Microscopy:

Cells with a fibrillar appearance in monolayers in FI medium are knownto become round after 1 week of culture in BIT medium. The cellscultured in monolayers (corresponding to the control) changed appearanceafter changing to BIT medium, indicating that the change in culturemedium indeed induced a change in the behaviour of the cells.

In the hydrogels (structures M1 and M2), the cells were primarily roundat the end of the amplification phase and continued to be round duringthe entire phase for the production of extracellular matrix. This pointis illustrated in particular in FIG. 7 .

At the end of culture, D35, the cells were all round in the hydrogels(structures M1 and M2), less so in monolayers.

In the three-dimensional structures, the cells could agglutinate theparticles of hydrogel and form a kind of “bead” with a compact form to agreater or lesser extent. This observation was made under the majorityof conditions containing the hydrogels, but not in the monolayers,however, which acted as the control. This observation constitutes aproof of the strong production of extracellular matrix which hadaccumulated around the cells. The cells produced more extracellularmatrix in a three-dimensional environment than in monolayers.

It should be noted that under certain conditions, however, thecomposition constituted by said beads remained injectable despite thesynthesis of a large quantity of extracellular matrix. Whatever thecase, the composition was implantable.

PCR Tests:

PCR was used to quantify the degree of transcription of the followingproteins: COLI, COLII and GAPDH. The degree of transcription acts as areference for comparing the levels of transcription of COLI and COLII.In fact, it is well known that the chondrocyte phenotype and theproduction of extracellular matrix are accompanied by a strong synthesisof COLII transcripts, while the COLI transcripts generally accompany theprocess of dedifferentiation, in particular into a fibroblast phenotype.The results are presented in FIG. 8 .

The COLII/GAPDH results produced at the end of the ECM synthesis step,show that there are more COLII transcripts for the cultures in hydrogelthan for the monolayer cultures. The COLI/GAPDH results show that, incontrast, there are more COLI transcripts in the monolayer cultures thanin the cultures within the three-dimensional structures.

The result of the calculation of the COLII/COLI ratio is illustrated inFIG. 9 . It shows that the ratio is indeed better, and in fact thatre-differentiation is much better after dedifferentiation within the 3Dstructures constituted by particles of chitosan hydrogel compared withthe monolayers.

The three-dimensional environment tested thus substantially favours there-differentiation of dedifferentiated chondrocytes following priorintense multiplication, by a ratio of at least 6. This 3D structurefavours the expression of chondrocyte phenotype.

The culture conditions (three-dimensional structure based on chitosanhydrogel, and BIT culture medium) thus favour the re-differentiation ofchondrocytes and the production of cartilaginous matrix, compared withmonolayer culture.

Western Blot Tests:

After having verified the degrees of transcription of the ColI and ColIIgenes as a function of the culture conditions (3D or monolayers), thedegree of synthesis of the corresponding proteins was verified using theWestern blot technique. The results of the various Western blots areillustrated in FIG. 10 .

The results for the anti-COLII Western blot revealed the presence ofCOLII in all conditions. The results for the anti-COLI WB revealed moreintense spots in monolayers. This observation corroborates the factrevealed in Q-PCR: the COLII/COLI ratio is higher in 3D hydrogelstructures than in monolayers.

The Western blot analysis showed the expression of characteristicproteins of articular cartilage in the 3D structure.

Immunohistochemistry Results:

The compositions obtained were then observed using immunohistochemistryin order to compare the implementation of the novel method using thethree-dimensional structure, and the traditional culture usingmonolayers, at the level of the synthesis of ECM, proteoglycans, andcollagen types I and II. The results are illustrated in the photos ofFIG. 11 .

The presence of more proteoglycans in the three-dimensional structures(M1 and M2 in FIG. 11 ) than in the monolayers (MC in FIG. 11 ) areclearly observed because of the Safranin O staining (SO), whichdemonstrates the presence of GAG. Furthermore, the production of a lotof extracellular matrix and type II collagen was observed on theimmunohistochemistry images for cells in the three-dimensionalenvironment respectively evidenced with HES staining, which demonstratesthe presence of nuclei and ECM, and by collagen II immunolabelling,which demonstrates the presence of COLII. Highlighting of the cells inthe matrix by collagen I immunolabelling also confirms the quasi-absenceof collagen I when the chondrocytes are cultured in thethree-dimensional structures.

Example 6: Chondrocytes Implantation

The assembly of the cells and the 3D structure (i.e. either thestructure M1 constituted by particles of chitosan hydrogel or thestructure M2, constituted by particles of chitosan hydrogel supplementedwith an anionic molecule like hyaluronic acid) constitutes, at the endof culture, i.e. between 3 and 6 weeks, a cartilaginous neo-tissue whichmay be injected or implanted by arthroscopy.

It is clearly possible to increase the number of cells in an insert byincreasing the quantity of hydrogel, keeping however the same conditionsfor the number of cells with respect to the mass of hydrogel or thenumber of hydrogel particles constituting the 3D structure.

By way of example, for a sample of 0.3 g-0.5 g of human cartilage,1-1.5×10⁶ cells (chondrocytes) may be extracted. Since in the precedingexamples the inventors have demonstrated that, starting from 6×10⁵initial cells per insert, it is possible to obtain from them 3.6×10⁶cells/insert in 0.09 g of hydrogel structure, corresponding to 80-84particles of hydrogel, the following concentration data were obtained:

-   -   initial concentration of 6.7×10⁶ cells/g of biomaterial,    -   final concentration of more than 40×10⁶ cells/g of biomaterial        (3D structure).

For a sample from 1 to 1.5×10⁶ cells, then, more than 3×10⁶ cells can beobtained, or even more than 10.5×10⁶ cells at the end of the method,after 2-5 weeks, which is amply sufficient for a construct where therecommended quantities are 3.2-6.5×10⁶ cells.

In conclusion for the preceding examples, the following points areobserved: During the amplification/multiplication phase:

-   -   an equivalent yield or in fact a greater yield in the        three-dimensional structure comprising particles of pure        chitosan hydrogel compared with monolayer culture,    -   a much higher yield in three-dimensional structure comprising        hydrogel particles supplemented with hyaluronic acid than in        monolayers.

During the phase for differentiation and ECM production:

-   -   a re-differentiation of cells inside 3D structures and in        monolayers, as proved by the PCR, WB and immunohistochemistry        analyses;    -   a ratio of COLII/COLI messenger RNA which is significantly        higher in the three-dimensional structure compared with        monolayer culture,    -   a COLII/COLI protein ratio which is significantly higher in        three-dimensional structure than in monolayers,    -   a stable chondrocyte phenotype in the 3D structure,    -   abundant production of cartilaginous matrix in the 3D structure.

The succession of steps in the same three-dimensional medium comprisingparticles of chitosan hydrogel with/without structuring molecule,amplification then differentiation/chondrogenesis is highly favourableto the production of an injectable or implantable cartilaginousneo-tissue with excellent mechanical and biological properties.

The addition of hyaluronic acid improves the system by accelerating theprocess for the amplification of cells and meaning that the number ofcells to be implanted can be increased, or a week can be saved over theoverall protocol.

The configuration of the structure can be used to optimize the contactsurface with the cells.

Example 7: Comparison Between Various 3D Structures

The inventors reproduced the 3D structures described in the precedingexamples, in particular in Example 2, by varying the type of chitosanconstituting the hydrogel particles, the size of the hydrogel particlesand the presence and the concentration of hyaluronic acid. Theproliferation ratios were compared and the results obtained areillustrated in Table 1. The value 1 was attributed to the structure M1corresponding to particles of several hundred microns obtained fromchitosan of fungi.

The proliferation ratio obtained with the structure M2 (extracted fromfungi and supplemented with 2M hyaluronic acid) was twice as high as thestructure M1 (extracted from fungi, not supplemented with hyaluronicacid), which itself makes it possible to obtain a proliferation rate 1.5times higher than chitosan extracted from squid, or in fact withparticles with a size of the order of tens of μm.

TABLE 1 _Proliferation ratios Fragments, Fragments, hundreds of μm tensof μm Chitosan extracted from  2** fungi supplemented with 2M HAChitosan extracted from 1.2 fungi supplemented with 1M HA Chitosanextracted from 1*  0.67 fungi Chitosan extracted from  0.67 squid*structure M1; **structure M2

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The invention claimed is:
 1. A three-dimensional structure formed byparticles of physical hydrogel comprising of chitosan or chitosanderivative only, wherein the particles are electrostaticallycross-linked by an anionic polymer, and wherein the anionic polymer isadded at the surface of said particles after gelling the hydrogel ofchitosan or chitosan derivative, and wherein said physical hydrogel issynthesized without a cross-linking agent.
 2. The three-dimensionalstructure according to claim 1, wherein said anionic polymer ishyaluronic acid, or hyaluronic acid derivative or a hyaluronic acidcomplex.
 3. The three-dimensional structure according to claim 1,wherein said particles of physical hydrogel have a mean size in therange of 400 μm to 700 μm.
 4. The three-dimensional structure accordingto claim 1, wherein said particles of physical hydrogel is made of purechitosan, and wherein said chitosan has a weight average molecularweight higher than 150 kDa.
 5. The three-dimensional structure accordingto claim 4, wherein the chitosan has a weight average molecular weightof 150 kDa to 220 kDa.
 6. The three-dimensional structure according toclaim 1, wherein the chitosan is extracted from fungi.
 7. Thethree-dimensional structure according to claim 6, wherein said particlesof physical hydrogel have a mean size in the range of 10 μm to 1.5 mm.8. The three-dimensional structure according to claim 1, wherein thewater content of said physical hydrogel of chitosan or chitosanderivative, is more than 70%.
 9. The three-dimensional structureaccording to claim 8, wherein said water content is more than 80%. 10.The three-dimensional structure according to claim 1, wherein thehyaluronic acid is extracted by bacterial fermentation with a molecularmass by weight of more than 1 MDa.
 11. The three-dimensional structureaccording to claim 10, wherein the hyaluronic acid has a molecular massby weight of more than 2 MDa.
 12. The three-dimensional structureaccording to claim 1, wherein the relative proportion of said anionicmolecule with respect to said physical hydrogel of chitosan or chitosanderivative is in the range of 1% to 10%.
 13. The three-dimensionalstructure according to claim 12, wherein the relative proportion of saidanionic molecule with respect to said physical hydrogel of chitosan orchitosan derivative is in the range 1% to 3%.
 14. The three-dimensionalstructure according to claim 1, wherein the cell viability of cellsseeded in the structure is more than 93% after 7 days seeding.
 15. Thethree-dimensional structure according to claim 1, wherein said structureensures an optimized spatial distribution of seeded cells.
 16. Athree-dimensional structure comprising fragments or particles ofphysical hydrogel comprising of chitosan or a chitosan derivative only,electrostatically cross-linked by an anionic molecule at the surface ofsaid particles after gelling the hydrogel of chitosan or chitosanderivative, and wherein said physical hydrogel is synthesized without across-linking agent, said structure being free from cells such that thethree-dimensional structure is precisely colonized in vivo by the cells.17. A method for culturing cells, comprising seeding cells in thethree-dimensional structure according to claim 1, wherein said cells donot penetrate into said particles of physical hydrogel, and wherein saidphysical hydrogel remains as support for implanted cells hereby becomingpart of the structure.
 18. The method according to claim 17, whereinsaid cells are selected from the group consisting in chondrocytes,primary cells differentiated into chondrocytes, precursors cells ofchondrocytes, induced pluripotent cells, mesenchymal stem cells andcombination thereof and wherein said cells are autologous or allogeniccells.
 19. The method according to claim 17, wherein said cells areselected from the group consisting in bone cells, fibroblasts,keratinocytes and combination thereof.
 20. The method according to claim17, wherein cells will proliferate and synthesize extracellular matrix.21. A method for treating a patient suffering from any diseasecharacterized by a degradation or disappearance of cartilaginous tissue,in particular cartilaginous defect, comprising the steps of: seedingcells in a three-dimensional structure according to claim 1, whereinsaid cells are chosen from the group consisting in chondrocytes, primarycells differentiated into chondrocytes, precursors cells ofchondrocytes, induced pluripotent cells, mesenchymal stem cells andcombination thereof; culturing and proliferating said seeded cells, thusproducing a cartilage gel, and implanting the cartilage gel thusobtained into the patient.